Los autores no tienen nada que revelar.

1. Preparación de cubreobjetos de vidrio para FCA <ol><li> Clean 12 mm cubreobjetos con toallitas bajos pelusa. </li><li> Llama el cubreobjetos mm 12 y el cubreobjetos de 14 mm en la cúpula de cada 35 mm de vidrio plato inferior pasándolos a través de un mechero Bunsen con unas pinzas. </li><li> Tratar los micropocillos con 200 l de NaOH 0,1 N durante 5 min a temperatura ambiente. </li><li> Aspirar y aire sequen los pocillos durante 30 minutos. </li><li> Tratar a los pocillos con 50 a 100 l de 3-aminopropiltrimetoxisilano durante 10 min a temperatura ambiente en la campana de humos. Este producto químico reacciona con el plástico; por lo tanto, utilizar pipetas de vidrio y no llene los pocillos completamente para evitar el contacto con el plástico plato. </li><li> Lavar los pocillos con agua ultrapura durante aproximadamente 10 min hasta que el 3-aminopropiltrimetoxisilano se vuelve claro. </li><li> Lavar los pocillos con agua ultrapura dos veces utilizando un frasco. </li><li> Lavar los pocillos con 2 ml de agua ultrapura unt temperatura ambiente en un agitador ajustado a una velocidad media (~ 1 Hz) durante 10 min. </li><li> Aspirar y aire sequen los pocillos durante 30 minutos. </li><li> Tratar los pocillos con 2 ml de solución de glutaraldehído al 0,5% a temperatura ambiente en un agitador ajustado a una velocidad media (~ 1 Hz) durante 30 min. </li><li> Lavar los pocillos con 2 ml de agua ultrapura a temperatura ambiente en un agitador a velocidad media (~ 1 Hz) durante 10 min. Repetir dos veces más para un total de 30 min. </li><li> Pocillos secos en un ángulo empinado (60º o más) durante 30 minutos. Nota: Los platos se pueden almacenar durante 2 meses en un desecador. </li></ol> 2. Preparación de la FCA para invadopodios ensayos <ol><li> Para una solución de la PAA suave (8% de acrilamida y 0,05% BIS) 1 ml, combinar 200 l de 40% de acrilamida, 25 l de 2% BIS y 574 l de agua ultrapura (Tabla 1). </li><li> Para una solución de la PAA duro (8% de acrilamida y 0,35% BIS) 1 ml, combinar 200 l de 40% de acrilamida, 175 &amp; #181; l de 2% BIS, y 409 l de agua ultrapura (Tabla 1). </li><li> Para una solución de la PAA rígido (12% de acrilamida y 0,60% BIS) 1 ml, combinar 300 l de 40% de acrilamida, 300 l de 2% BIS, y 169 l de agua ultrapura (Tabla 1). </li><li> Desgasificar las soluciones para 15 min. Añadir 200, 215, y 230 l de 1 mg / ml de fibronectina de plasma humano en agua ultrapura a las soluciones de PAA blandos, duros, y rígidas, respectivamente. Para todas las soluciones, añadir 1 l de / ml de éster de ácido acrílico N-hidroxisuccinimida 10 mg (NHS), 5 l de un persulfato de amonio 100 mg / ml, y 2 l de N, N, N &#39;, N&#39;-tetrametiletilendiamina (TEMED ; Tabla 1). Mezclar con pipeteo suave y evitar la creación de burbujas en soluciones. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> PAA (1 ml) </td><td> 40% AA (L) </td><td> 2% (L) </td><td> Ultrapura Agua (L) </td><td> 1 mg / ml FN (L) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 mg / ml NHS Ester (L) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 mg / ml APS (L) </td><td> TEMED (L) </td><td> Elástico Módulo (Pa) </td></tr><tr><td> Suave </td><td> 200 </td><td> 25 </td><td> 574 </td><td> 200 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 1023 </td></tr><tr><td> Duro </td><td> 200 </td><td> 175 </td><td> 409 </td><td> 215 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 7307 </td></tr><tr><td> Rígido </td><td> 300 </td><td> 300 </td><td> 169 </td><td> 230 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 22692 </td></tr></tbody></table> Tabla 1: Ingrediente volumes y que resulta módulos elásticos para FCA blandos, duros y rígidos utilizados en los ensayos invadopodios. AA = acrilamida, FN = fibronectina, APS = persulfato de amonio. <ol start="5"><li> Pipetear 8,48 l de la solución de PAA en el centro de cada pocillo. Nota: Este volumen se obtiene teóricamente un PAA de 75 micras de espesor. </li><li> Baje suavemente el lado flameado del cubreobjetos de 12 mm a la gota en el pocillo, en pinzas. </li><li> Deje que la solución PAA intercalada para polimerizar durante 15-30 minutos. Verifique polimerización marcando la solución sobrante. </li><li> Añadir 2 ml de 1x PBS a cada placa y retirar los cubreobjetos de 12 mm con pinzas. Nota: 10x PBS se produce en el laboratorio y compuesta por 0.011 M KH 2 PO 4, NaCl 1,54 y 0,056 M Na 2 HPO 4. </li><li> Lavar los pocillos con 2 ml de PBS 1x a temperatura ambiente durante 5 min. Repetir dos veces más para un total de 15 min. </li></ol> 3. Preparación de FCA para la tracción de la Fuerza ensayos <ol><li> Para una solución de la PAA suave (8% de acrilamida y 0,05% BIS) 1 ml, combinar 200 l de 40% de acrilamida, 25 l de 2% BIS y 566 l de agua ultrapura (Tabla 2). </li><li> Para una solución de la PAA duro (8% de acrilamida y 0,35% BIS) 1 ml, combinar 200 l de 40% de acrilamida, 175 l de 2% BIS, y 401 l de agua ultrapura (Tabla 2). </li><li> Para una solución de la PAA rígido (12% de acrilamida y 0,60% BIS) 1 ml, combinar 300 l de 40% de acrilamida, 300 l de 2% BIS, y 161 l de agua ultrapura (Tabla 2). </li><li> Desgasificar las soluciones para 15 min. Añadir 200, 215, y 230 l de 1 mg / ml de fibronectina de plasma humano a las soluciones de PAA blandos, duros, y rígidas, respectivamente. Sonicar 8 l de 200 nm microesferas rojas fluorescentes (excitación / emisión de 580/605 nm) durante 30 segundos. Para cada solución, añadir 8 l de 200 microesferas fluorescentes rojas nm,1 l de ácido acrílico 10 mg / ml de éster de NHS, 5 l de una persulfato de amonio 100 mg / ml, y 2 l de TEMED (Tabla 2). Mezclar con pipeteo suave y evitar la creación de burbujas en soluciones. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td style="width:40px"> PAA (1 ml) </td><td> 40% AA (L) </td><td> 2% BIS (L) </td><td style="padding-left: 4px; padding-right: 4px;"> Ultrapura Agua (L) </td><td> 1 mg / ml FN (L) </td><td> Micro-esferas (L) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 mg / ml NHS Ester (L) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 mg / ml APS (L) </td><td> TEMED (L) </td><td> Elástico Módulo (Pa) </td></tr><tr><td> Suave </td><td> 200 </td><td> 25 </td><td> 566 </td><td> 200 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 1023 </td></tr><tr><td> Duro </td><td> 200 </td><td> 175 </td><td> 401 </td><td> 215 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 7307 </td></tr><tr><td> Rígido </td><td> 300 </td><td> 300 </td><td> 161 </td><td> 230 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 22692 </td></tr></tbody></table> Tabla 2:. Volúmenes de ingredientes y resultando módulos elásticos para FCA blandos, duros y rígidos utilizados en los ensayos de la fuerza de tracción AA = acrilamida, fn = fibronectina, APS = persulfato de amonio. <ol start="5"><li> Pipetear 8,48 l de la solución de PAA en el centro de cada pocillo. Nota: Este volumen se obtiene teóricamente un PAA de 75 micras de espesor. </li><li> Baje suavemente el lado flameado del ªe 12 mm cubreobjetos sobre la gota en el pocillo, en pinzas. </li><li> Deje que la solución PAA intercalada para polimerizar durante 15-30 minutos. Verifique polimerización comprobando solución sobrante. </li><li> Añadir 2 ml de 1x PBS a cada placa y retirar los cubreobjetos de 12 mm con pinzas. </li><li> Lavar los pocillos con 2 ml de PBS 1x a temperatura ambiente durante 5 min. Repetir dos veces más para un total de 15 min. </li></ol> 4. Preparación de ECM para invadopodios ensayos <ol><li> Calentar la solución de gelatina (gelatina al 1% y 1% de sacarosa) a 37 ° C. </li><li> Tratar los FCA con 150 l de la solución de gelatina durante 1 min luego cuidadosamente aspirado desde el fondo de los pocillos al sostener los platos con fondo de cristal en un ángulo de 45 °. </li><li> Se seca la capa fina restante de gelatina sobre las DAP en los micropocillos en un ángulo empinado (60 ° o mayor) para optimizar el secado durante 60 min. </li><li> Tratar los pocillos con 2 ml de una solución fría de glutaraldehído 0,5% en iCE para 15 min seguido por temperatura ambiente durante 30 min. </li><li> Lavar los pocillos con 2 ml de PBS 1x para una 5 min. Repetir dos veces más para un total de 15 min. </li><li> Tratar los pocillos con 2 ml de una solución de borohidruro de sodio durante 1 min. Golpee suavemente los platos en el banco para eliminar las burbujas que se forman en la superficie de la gelatina. </li><li> Aspirar y lavar los pocillos con 2 ml de PBS 1x durante 5 min. Repetir dos veces más para un total de 15 min. </li><li> Se diluye una solución de fibronectina de plasma humano marcado con FITC a 50 g / ml con un PBS 1X y se centrifuga a 175.000 xg a 4 ° C durante 15 min. Cualquiera de adquirir comercialmente disponible fibronectina etiquetado o la etiqueta de la siguiente manera: <ol><li> Disolver 5 mg de fibronectina en 10 ml de tampón de borato (metaborato de sodio tetrahidratado 170 mM y NaCl 40 mM) y el lugar en tubos de diálisis. </li><li> Etiqueta de la fibronectina mediante la colocación de la tubería en 200 ml de tampón de borato que contiene 6 mg de FITC disuelto a temperatura ambiente en un agitador magnéticofijado en la posición más baja de 1,5 horas en la oscuridad. </li><li> Se dializa con 500 ml de una PBS 1x en un agitador magnético fijado en la posición más baja durante 3 días en una habitación oscura y fría (4 ° C) con dos volúmenes cambia un día. </li><li> La concentración de fibronectina marcada con FITC calcular sobre la base de la densidad óptica de alícuotas diluidas (1: 200) a 280 nm y 493 nm como [OD 280 - (0,36 x OD 493)] / 1,4 veces el factor de dilución de 200 veces 2 (que representa para el glicerol concentración de la fibronectina en el paso siguiente) resulta en unidades de mg / ml. </li><li> Dializar noche en una solución de glicerol al 50% en un agitador magnético fijado en la posición más baja durante la noche en una habitación fría oscuridad a 4 ° C.) </li></ol></li><li> Tratar los micropocillos con 150 l de la solución de fibronectina marcada con FITC a temperatura ambiente durante 60 min en la oscuridad. </li><li> Aspirar cuidadosamente la solución de fibronectina marcada con FITC desde el fondo de los pocillos al sostener los platos con fondo de cristal enun ángulo de 45 ° y luego se llenan los platos inferiores de vidrio con solución de etanol al 70% a temperatura ambiente durante 10 min en una campana de cultivo de células oscuro para la esterilización. También llenar tapas de los recipientes de vidrio de fondo o limpiar con toallitas bajos pelusa empapado en etanol al 70%. </li><li> Lave los platos con fondo de cristal, llenándolos con 1x PBS durante 5 min. Repetir dos veces más con 2 ml de PBS 1x para un total de 15 min. </li></ol> 5. Preparación e Imagen de invadopodios ensayos <ol><li> Añadir 25,000 células cancerosas en 2 ml de medio invadopodios (1: 1 DMEM y medio RPMI 1640 con 5% de suero baja en proteínas, suero bovino fetal al 10%, y 20 ng / ml de factor de crecimiento epidérmico recién añadido) a los platos de fondo de cristal y incubar durante la noche. </li><li> Aspirar y tratar las microcubetas con 2 ml de una solución de paraformaldehído al 3,7% a temperatura ambiente en la oscuridad durante 20 min para fijar las células. </li><li> Después de dos lavados rápidos con 2 ml de PBS 1x, tratar los micropocillos con 2 ml de una solución de 0,1% de Triton X-100 en tempe habitaciónratura en la oscuridad durante 5 min para permeabilizar las células. </li><li> Después de un lavado rápido con 2 ml de PBS 1x, tratar las microcubetas con 3% de albúmina de suero bovino solución de bloqueo a temperatura ambiente en la oscuridad durante 60 min para bloquear las células. </li><li> Incubar los pocillos con 150 l de anticuerpo de ratón anti-cortactin (1: 750) en solución de bloqueo a temperatura ambiente en la oscuridad durante 60 min. </li><li> Lavar los pocillos con 2 ml de PBS 1x para 5 min. Repetir dos veces más para un total de 15 min. </li><li> Incubar los pocillos con 150 l de anticuerpo de cabra anti-ratón 633 de anticuerpos (1: 500) y 546 faloidina (1: 750) en solución de bloqueo a temperatura ambiente en la oscuridad durante 60 min. </li><li> Lavar los pocillos con 2 ml de PBS 1x para 5 min. Repetir dos veces más para un total de 15 min. </li><li> Añada seis gotas de medio de montaje para llenar cada pocillo y cubra con 22 x 22 cubreobjetos. </li><li> Cortactin Imagen y actina para identificar invadopodios utilizando filtros de excitación y emisión de 632/647 nm y556/570 nm, respectivamente, utilizando una lente de inmersión en aceite de alta NA, 40X en un microscopio invertido de campo amplio. Del mismo modo, la imagen de fibronectina para identificar la degradación de ECM utilizando un filtro de excitación y emisión de 492/518 nm. </li></ol> 6. Preparación e Imagen de la fuerza de tracción Ensayos <ol><li> Añadir 15,000 células cancerosas en 2 ml de medio invadopodios a los platos con fondo de cristal y se incuba durante la noche. </li><li> Aspirar medio en los platos de fondo de cristal y reemplazar con 2 ml de medio L-15, con los mismos suplementos (5% de suero baja en proteínas, suero bovino fetal al 10%, y 20 ng / ml de factor de crecimiento epidérmico recién añadido), pre -warmed a 37 ° C. </li><li> Incubar los platos inferiores de vidrio en una cámara ambiental en un microscopio invertido pre-equilibrada a 37 ° C con alta humedad durante 1 hr. </li><li> Tome cuatro conjuntos de imágenes con una alta NA, 40X lente de un microscopio de campo amplio invertida con una etapa automatizado para marcar posiciones celulares: <ol><li> Celdas de imagen sobre dep de los PAA utilizando contraste de fase (&quot;fase&quot; de la imagen). </li><li> Image microesferas fluorescentes utilizando filtros de excitación y emisión de 560/645 nm, centrándose ligeramente por debajo de las células hasta que la primera capa de microesferas en la parte superior de la FCA entra en el foco (&quot;estresados&quot; imagen). </li><li> Además, se centran en la parte inferior de la FCA (utilizando las microesferas como marcadores) y tomar una imagen (imagen &quot;de abajo&quot;). Nota: El propósito de esta imagen es para registrar la posición z para obtener el espesor PAA real en cada posición para el cálculo de la fuerza de tracción como se describe en resultados representativos. </li><li> Después de estas imágenes han sido adquiridos en múltiples posiciones, mezclar suavemente en 220 l de solución al 10% de Triton-X para eliminar las células. Microesferas de la imagen en la parte superior de la FCA en cada posición como se describe anteriormente (imagen &quot;nulo&quot;). </li></ol></li></ol>

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	<li>Paszek, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer cell. 8, 241-254 (2005).</li><li>Jaalouk, D. E., Lammerding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+gone+awry.">Mechanotransduction gone awry.</a> Nature. 10, 63-73 (2009).</li><li>Paszek, M. J., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tension+mounts:+mechanics+meets+morphogenesis+and+malignancy.">The tension mounts: mechanics meets morphogenesis and malignancy.</a> Journal of mammary gland biology and neoplasia. 9, 325-342 (2004).</li><li>Parekh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+and+modulation+of+invadopodia+across+a+wide+range+of+rigidities.">Sensing and modulation of invadopodia across a wide range of rigidities.</a> Biophysical. 100, 573-582 (2011).</li><li>Jerrell, R. J., Parekh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+mediate+extracellular+matrix+degradation+by+invadopodia.">Cellular traction stresses mediate extracellular matrix degradation by invadopodia.</a> Acta biomaterialia. 10, 1886-1896 (2014).</li><li>Kraning-Rush, C. M., Califano, J. P., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+increase+with+increasing+metastatic+potential.">Cellular traction stresses increase with increasing metastatic potential.</a> PLoS ONE. 7, e32572 (2012).</li><li>Haage, A., Nam, D. H., Ge, X., Schneider, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase-14+is+a+mechanically+regulated+activator+of+secreted+MMPs+and+invasion.">Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion.</a> Biochemical and biophysical research communications. , (2014).</li><li>Haage, A., Schneider, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+contractility+and+extracellular+matrix+stiffness+regulate+matrix+metalloproteinase+activity+in+pancreatic+cancer+cells.">Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.</a> FASEB J. , (2014).</li><li>Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia:+specialized+cell+structures+for+cancer+invasion.">Invadopodia: specialized cell structures for cancer invasion.</a> Clinical &amp; experimental metastasis. 23, 97-105 (2006).</li><li>Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia.">Invadopodia.</a> Curr Biol. 18, R362-364 (2008).</li><li>Bravo-Cordero, J. J., Hodgson, L., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+cell+invasion+and+migration+during+metastasis.">Directed cell invasion and migration during metastasis.</a> Current opinion in cell biology. 24, 277-283 (2012).</li><li>Alexander, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+rigidity+promotes+invadopodia+activity.">Extracellular matrix rigidity promotes invadopodia activity.</a> Curr Biol. 18, 1295-1299 (2008).</li><li>Barlow, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+breast+cancer+risk+prediction+model+for+women+undergoing+screening+mammography.">Prospective breast cancer risk prediction model for women undergoing screening mammography.</a> Journal of the National Cancer Institute. 98, 1204-1214 (2006).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projecting+absolute+invasive+breast+cancer+risk+in+white+women+with+a+model+that+includes+mammographic+density.">Projecting absolute invasive breast cancer risk in white women with a model that includes mammographic density.</a> Journal of the National Cancer Institute. 98, 1215-1226 (2006).</li><li>Butcher, D. T., Alliston, T., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tense+situation:+forcing+tumour+progression.">A tense situation: forcing tumour progression.</a> Nature reviews. Cancer. 9, 108-122 (2009).</li><li>Zaman, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+tumor+cells+in+3D+matrices+is+governed+by+matrix+stiffness+along+with+cell-matrix+adhesion+and+proteolysis.">Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 10889-10894 (2006).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+density-induced+mechanoregulation+of+breast+cell+phenotype,+signaling+and+gene+expression+through+a+FAK-ERK+linkage.">Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage.</a> Oncogene. 28, 4326-4343 (2009).</li><li>Munevar, S., Wang, Y., Dembo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+force+microscopy+of+migrating+normal+and+H-ras+transformed+3T3+fibroblasts.">Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts.</a> Biophysical journal. 80, 1744-1757 (2001).</li><li>Rosel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+Rho/ROCK+signaling+in+sarcoma+cells+drives+invasion+and+increased+generation+of+protrusive+forces.">Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.</a> Mol Cancer Res. 6, 1410-1420 (2008).</li><li>Indra, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+correlation+of+mechanical+forces+and+metastatic+capacity.">An in vitro correlation of mechanical forces and metastatic capacity.</a> Phys Biol. 8, 015015 (2011).</li><li>Parekh, A., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+invasiveness+by+the+physical+extracellular+matrix+environment.">Regulation of cancer invasiveness by the physical extracellular matrix environment.</a> Cell adhesion &amp; migration. 3, 288-292 (2009).</li><li>Weaver, A. M., Page, J. M., Guelcher, S. A., Parekh, A., Coutts, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology in Adhesion Protein Protocols. 1046, 171-189 (2013).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples).">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples).</a> Physics in medicine and biology. 52, 1565-1576 (2007).</li><li>Wang, J. H., Lin, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

Este trabajo fue apoyado por el Instituto Nacional del Cáncer de los Institutos Nacionales de Salud, bajo el premio número K25CA143412 (Parekh). Nos gustaría reconocer que el apoyo adicional fue proporcionada por el Departamento de Otorrinolaringología. El contenido es responsabilidad exclusiva de los autores y no representa necesariamente las opiniones oficiales de los Institutos Nacionales de Salud.

Se presenta un método para la fabricación de FCA que se pueden utilizar como la base para los ensayos de invadopodios y fuerza de tracción para correlacionar comportamientos celulares invasivas y contráctiles. Mientras FCA han sido utilizados para observar los efectos de rigidez en las células y calcular las fuerzas de tracción 18,24,27, este protocolo es el primero en desarrollar ensayos paralelos basados ​​en APA con las mismas rigideces correlacionar comportamientos celulares invasivos y contrác...

La rigidez de la ECM asociado al tumor ha sido identificado como un factor significativo en la conducción de comportamiento maligno mediante el aumento de la contractilidad actomyosin 1-3. Mientras que este efecto se ha demostrado principalmente con células de cáncer de mama, la rigidez de la matriz se ha encontrado para alterar las propiedades invasivas de las células derivadas de una variedad de cánceres 4-8 lo que sugiere que la rigidez tumor pueden desempeñar un papel en otro tipo de cánceres. Para penetrar en los tejidos entrecruzados durante la migración invasivo, las células cancerosas utilizan protuberancias adhesivas actina-ricos conocidos como invadopodios que localizar proteinasas para degradar focalmente el ECM 9. Invadopodios se consideran una característica de las células invasoras y han sido implicados en la invasión de células tumorales y la metástasis 10,11. El trabajo previo ha mostrado que la rigidez de la matriz puede regular los números invadopodios y la degradación de ECM asociado 4,12 través de la miosina II actividad y proteínas mechanosensitive 12. Dado quecorrelación entre la densidad del tumor y la agresividad del cáncer 13,14, estos resultados sugieren un mecanismo por el cual las células cancerosas pueden responder a los tejidos tumorales rígidos para conducir la invasión y la metástasis a través de la contractilidad actomiosina. En rigidez ECM vitro y en la densidad del tejido vivo se ha demostrado para regular el comportamiento invasivo de las células cancerosas 1,15-17. Mientras que la contractilidad actomyosin parece ser importante en este proceso, la corriente de conflicto estudios en cuanto a si la capacidad metastásica se correlaciona con aumento o disminución de las fuerzas contráctiles 6,18-20. Además, aún se desconoce si estas fuerzas median directamente la actividad invadopodios 21. Recientemente, hemos encontrado que las fuerzas contráctiles célula cancerosa dependían de la rigidez de la matriz y fueron predictivos de la degradación de ECM por invadopodios 5. Estos resultados sugieren que las fuerzas celulares pueden desempeñar un papel importante en la progresión del cáncer mediando invadopodios actividad en respuesta a las propiedades mecánicas del microambiente del tumor. Para correlacionar propiedades invasivas y contráctiles de las células cancerosas 5, modificamos un protocolo para la creación de FCA con diferentes rigideces que se utilizó anteriormente para investigar la actividad invadopodios rigidez dependiente 4,12,22. Por reticulación químicamente fibronectina plasmática humana en todo el FCA, estos hidrogeles modificados se pueden utilizar como la base para tanto invadopodios y los ensayos de fuerza de tracción para asegurar que las células experimentaron las mismas rigideces en ambos experimentos 5. En los ensayos de invadopodios, la fibronectina proporciona un dominio de unión natural para la gelatina para vincular el ECM superpuesto a la FCA para detectar degradación de la matriz. En los ensayos de fuerza de tracción, la fibronectina proporciona un ligando para la adhesión celular directa para detectar desplazamientos de microesferas utilizadas para calcular las fuerzas de tracción celulares. Este método resulta en lo que hemos llamado blando, duro,y PAA rígidos que están unidos a los platos con fondo de cristal y tienen módulos de elasticidad, E, de 1.023, 7.307 y 22.692 Pa 5 que abarcan la gama de propiedades mecánicas reportados para los tejidos normales y cancerosos 23.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-Aminopropyltrimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-Rad</td>
			<td>161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic acid NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>A8060</td>
			<td>prepare fresh in fume hood 10 mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Life Technologies</td>
			<td>A22283</td>
			<td>can also use rhodamine</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>prepare fresh 10% solution in 1X PBS</td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>18606</td>
			<td>use six drops to fill microwells</td>
		</tr>
		<tr>
			<td>BIS (2%)</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>RPI</td>
			<td>A30075</td>
			<td>make 3% for blocking solution in 1X PBS and store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips (12 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9777</td>
			<td>pre-equilibrate in borate buffer for 15-30 min</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>use to make acrylic acid NHS ester solution</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PHARMCO-AAPER</td>
			<td>E200</td>
			<td>dilute with ultrapure water to 70%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>SH30070.03</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Polysciences</td>
			<td>00639</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (35 mm coverslips)</td>
			<td>MatTek</td>
			<td>P35G-0-14-C</td>
			<td>coverslips are uncoated</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%)</td>
			<td>Polysciences</td>
			<td>01909</td>
			<td>dilute with 1X PBS to 0.5%</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa Fluor 633 antibody&#160;</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Life Technologies</td>
			<td>33016-015</td>
			<td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>PX-1565-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>mouse anti-cortactin 4F11 antibody&#160;</td>
			<td>EMD Millipore</td>
			<td>05-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>SX-0720-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23020</td>
			<td>use to make 10X PBS stock and borate buffer</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>dilute with ultrapure water to 0.1 N</td>
		</tr>
		<tr>
			<td>Nu-Serum (low-protein serum)</td>
			<td>BD Biosciences</td>
			<td>355500</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros</td>
			<td>416785000</td>
			<td>typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td>
		</tr>
		<tr>
			<td>PBS (sterile)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>use for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Cellgro</td>
			<td>10-040-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452882</td>
			<td>prepare fresh in fume hood 1 mg/ml in 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>sodium metaborate tetrahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0251</td>
			<td>use to make borate buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td>make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining</td>
		</tr>
	</tbody>
</table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

Tejidos tumorales rígidos han sido fuertemente implicados en la regulación de la migración celular y la invasión del cáncer. La migración invasiva a través de tejidos reticulados se ve facilitada por los salientes de actina-ricos llamados invadopodios que proteolíticamente degradan la matriz extracelular (ECM). Actividad invadopodios ha demostrado ser dependiente de la rigidez ECM y célula de cáncer de fuerzas contráctiles que sugieren que las señales de rigidez pueden regular estas estructuras subcelulares a través de la contractilidad actomiosina. Propiedades invasivas y contráctiles de las células cancerosas pueden ser correlacionados in vitro utilizando ensayos invadopodios y de la fuerza de tracción sobre la base de geles de poliacrilamida (PAA) de diferentes rigideces. Propiedades invasivas y contráctiles de las células cancerosas pueden ser correlacionados in vitro utilizando ensayos invadopodios y de la fuerza de tracción sobre la base de geles de poliacrilamida (PAA) de diferentes rigideces. Aunque existen algunas variaciones entre los dos ensayos, el protocolo presentado aquí proporciona un método para crear FCA ésimoa se puede utilizar en ambos ensayos y son fácilmente adaptables a las necesidades biológicas y técnicas específicas de los usuarios.

En el ensayo de invadopodios, invadopodios se identifican típicamente por colocalización de los marcadores como la actina y la cortactin en estructuras punteadas dentro del cuerpo de la célula (Figura 1). Tanto la degradación de forma activa y no degradante invadopodios puede ser contado y se diferencian por si estas estructuras están colocalized con áreas negras que carecen de señal fluorescente en la fibronectina marcada con FITC (Figura 1). Invadopodios se cuentan manualmente,...

Watch this Scientific Journal Video about Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells at JoVE.com

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigidez mecánica en el microambiente tumoral juega un papel fundamental en el impulso de comportamiento maligno mediante el aumento de la actividad invadopodios y actomyosin contractilidad. Geles de poliacrilamida utilizando (PaaS), ensayos de invadopodios y de la fuerza de tracción se pueden utilizar para estudiar las propiedades invasivas y contráctiles de las células cancerosas en respuesta a la matriz rigidez.

Los geles de poliacrilamida para invadopodios y Fuerza de tracción Ensayos sobre las células cancerosas

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

polyacrylamide-gels-for-invadopodia-traction-force-assays-on-cancer

Research

JoVE Journal

Medicine

12.0K vistas.  Vanderbilt University Medical Center.  Rigidez mecánica en el microambiente tumoral juega un papel fundamental en el impulso de comportamiento maligno mediante el aumento de la actividad invadopodios y actomyosin contractilidad. Geles de poliacrilamida utilizando (PaaS), ensayos de invadopodios y de la fuerza de tracción se pueden utilizar para estudiar las propiedades invasivas y contráctiles de las células cancerosas en respuesta a la matriz rigidez. 

Video: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Procesamiento de tejido primario Tumor cerebral para ensayos con células madre y flujo de Ordenación

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Investigación

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

La formación del epitelio prostático humano utilizando tejido recombinación de roedores Urogenital mesénquima seno y células madre humanas

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptación de semiautomatizado de células tumorales circulantes (CTC) Ensayos para aplicaciones clínicas y de investigación preclínica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Perfiles de microARN regulados por estrógeno en las células del cáncer de mama

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Método para la obtención de las células del cáncer de ovario primarios a partir de muestras sólidas

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

El enriquecimiento de las células madre quimiorresistente cáncer ovárico de líneas celulares humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Un Sistema organotípicos de alto rendimiento para la Caracterización de la sensibilidad a los fármacos de las células del mieloma múltiple primaria

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...